cells overexpressing rap2a  (Addgene inc)

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: ( A, B) Analysis of GBM patient versus control samples focused on the level of expression of human homologs of Drosophila ACD regulators by Hierarchical clustering ( A ) and the Gene Distance Matrix ( B ). Color-coded scale bar indicates fold level of expression. (C) Kaplan-Meier survival curves corresponding to patients in the TCGA (IDHwt) and Gravendeel (IDHwt) cohort in GBM datasets. Low RAP2A expression levels in human GBM are associated with a poor prognosis.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: (A) Different GBM cell lines show similar RAP2A mRNA levels and significantly lower levels than in control Astros. (B) RAP2A mRNA levels are significantly higher in the GB5 line after infecting this line with RAP2A (GB5-RAP2A). Data shown in the scaled bar graphs in A and B was analyzed with an ANOVA and a T-test, respectively; error bars show the SD; n= 2 (in A) and 3 (in B) different experiments. (C) Immunofluorescences of the GSC stem cell markers CD133, SOX2 and Nestin reveal a significant reduction in the protein intensity in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the box plots was analyzed with a U Mann-Whitney for CD133 and Nestin and with a T-test for Sox; the central lines represent the median and the box limits the lower and upper quartiles, as determined by R software; crosses represent sample means; error bars indicate the SEM; n= number of sample points; p values: * p <0.05, ** p <0.01, *** p <0.001, ns, not significant; scale bar: 10 μm.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Control, Expressing, MANN-WHITNEY, Software

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: RT-PCRs and Westerns blots show a significant decrease in the mRNA and protein expression levels, respectively, of the GSC markers CD133, SOX2 and Nestin in the GB5 RAP2A-expressing neurospheres (GB5-RAP2A) compared to the control neurospheres (GB5). Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s t test; error bars show the SD; n= 3 different experiments; p values: * p <0.05, ** p <0.01.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Expressing, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: (A) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significantly lower number of Ki67 expressing cells per neurosphere than control GB5 neurospheres. Data shown in the box plots was analyzed with a T-test; the central lines represent the median and the box limits the lower and upper quartiles, as determined by R software; crosses represent sample means; error bars indicate the SEM; n= number of sample points. (B) GB5 neurospheres expressing RAP2A (GB5-RAP2A) show a significant decrease in their size compared to control GB5 neurospheres of the same stage. Data shown in the scaled bar graphs was analyzed with an unpaired two-tailed Student’s T- test; error bars show the SD; n= total number of neurospheres of 3 different experiments; p values: * p <0.05; scale bars: 10 μm (in A) and 100μm (in B).

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Expressing, Control, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Human RAP2A Homolog of the Drosophila Asymmetric Cell Division Regulator Rap2l Targets the Stemness of Glioblastoma Stem Cells

doi: 10.1101/2025.01.28.635292

Figure Lengend Snippet: (A) Early stage GB5 neurospheres expressing RAP2A (GB5-RAP2A) show an odd number of cells significantly more frequently than control GB5 neurospheres, which show more frequently an even number of cells. (B) GB5-RAP2A dividing cells show a significant increase in the number of asymmetric NUMB localization in the progeny, compared to control GB5 dividing cells. Data shown in the bar graphs was analyzed with a Chi-Square test with Yates correction; n= number of neurosphere cell clusters analyzed. p values: ** p <0.01; scale bar: 20 μm.

Article Snippet: Lentiviral vectors were used to produce cells overexpressing RAP2A ( pLJM1RAP2A #Plasmid 19311, Addgene) or eGFP (pLJMEGFP #Plasmid 19319, Addgene) as infection control.

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Proton-Assisted Amino Acid Transporter PAT1 Complexes with Rag GTPases and Activates TORC1 on Late Endosomal and Lysosomal Membranes

doi: 10.1371/journal.pone.0036616

Figure Lengend Snippet: ( A , B ) Flag-PAT1 co-localises with a subset of the compartments containing endogenous RagC under both AA-starved (A) and AA-stimulated (B) conditions in a stable HEK-293 cell line overexpressing Flag-PAT1. ( C ) Under steady state conditions, immunoprecipitation of Flag-PAT1 leads to co-immunoprecipitation of endogenous RagC, but not tubulin. ( D ) Conversely, immunoprecipitation of Flag-RagD, but not Flag-Rap2A, both transiently expressed in HEK-293 cells, leads to co-immunoprecipitation of endogenous PAT1, but not tubulin, suggesting that the Rag GTPases complex with PAT1 in cells. Scale bar in A is 10 µm and applies to all panels in A and B.

Article Snippet: HEK-293 cells were transiently transfected one day after plating in 6-well plates using MATra transfection, as described in with Addgene plasmid 19316 (Flag pLJM1 RagD) or Addgene plasmid 19311 (Flag pLJM1 Rap2A) and incubated for two days prior to lysis.

Techniques: Immunoprecipitation